2. Description of Related Art
Our environment contains a multitude of microorganisms with which we are continuously interacting. These interactions can be beneficial, i.e., fermentations to produce wine, vinegar or antibiotics; neutral; or even harmful, as in the case of infectious diseases. The ubiquitous presence of these microorganisms, thus, creates a continuing need for the detection, identification and study of the presence and metabolic activity of such microorganisms.
While the science of microbiology has changed significantly in the last 25 years, many procedures for the detection, identification and analysis of the behavior of microorganisms are still time consuming. For example, in the area of antimicrobic susceptibility testing nearly half of all testing in hospitals in the United States still use the Bauer-Kirby Disc Method. This method uses the presence or absence of visible growth of the microorganisms to indicate the efficacy of an antimicrobic compound, and generally requires an 18 to 24 hour incubation period to allow for microorganism growth before a result can be obtained. A decrease in the time required to obtain such antimicrobic susceptibility information is needed.
Another popular method for antimicrobic susceptibility testing is the broth micro-dilution method, such as the Sceptor(copyright) System for identification and antimicrobic susceptibility testing or organisms (Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.). The system uses a disposable plastic panel having a plurality of low volume cupulas (ca. 0.4 ml per cupula), each containing a different test compound or a different concentration of a test compound dried on the cupula surface. The organism to be tested is suspended in the desired testing medium, and aliquots are delivered to the individual cupulas of the test panel. The reagent dried on the panel dissolves in the sample, and the system is then incubated overnight (18 to 24 hrs.) to allow sufficient time for the organisms to interact with the reagent and for visible growth to appear. The panel is subsequently examined visually for the presence or absence of growth, thereby obtaining information on the susceptibility of the organism undergoing testing. Additional wells aid in identifying the organism. However, this test method suffers from the drawback of also requiring a long incubation period.
One approach to the reduction of the incubation time is to monitor metabolic activity of the microorganisms, rather than growth of colonies. Many approaches have been reported in the attempt to rapidly and accurately monitor such metabolic activity.
For example, apparatus utilizing light scattering optical means have been used to determine susceptibility by probing the change in size or number of microorganisms in the presence of various antimicrobic compounds. Commercial instruments utilizing these principles are exemplified by the Vitec System (BioMerieux Corp.). This system claims to yield information on antimicrobic susceptibility of microorganisms within 6 hours for many organism and drug combinations. Other combinations can require as long as 18 hours before the antimicrobic susceptibility of the organism can be determined by this machine.
Additionally, modifications of the Bauer-Kirby procedure have been developed which allow certain samples to be read in four to six hours. However, such a system is xe2x80x9cdestructivexe2x80x9d in nature, requiring the spraying of a developing solution of a color forming dye onto the test plate. Re-incubation and reading at a later time is, thus, not possible and if the rapid technique fails, the experiment cannot be continued for a standard evaluation at a later time.
A bioluminescent method based on the quantity of ATP present in multiplying organisms has been described as yielding results of antimicrobic susceptibility testing in four and half hours for certain compositions (Wheat et al.). However, the procedure tends to be cumbersome and broad applicability has not been shown.
Other approaches have involved monitoring of microbial oxygen consumption by the measurement of pH and/or hemoglobin color change, or by the use of dyes such as triphenyltetrazolium chloride and resazurin, that change color in response to the total redox potential of the liquid test medium.
The monitoring of the consumption of dissolved oxygen by microorganisms, as a marker of their metabolism, has been studied for many years. For example, C. E. Clifton monitored the oxygen consumption of microorganisms over a period of several days using a Warburg flask in 1937. This method measured the change in oxygen concentration in a slow and cumbersome manner.
The xe2x80x9cClarkxe2x80x9d electrode, a newer electrochemical device, is also commonly used to measure dissolved oxygen. Unfortunately, the Clark electrode consumes oxygen during use (thereby reducing the oxygen available to the microorganisms) and the xe2x80x9cstandardxe2x80x9d size electrode is typically used only to measure volumes of 100 mls or greater to prevent the electrode from interfering with the measurements.
A xe2x80x9cminiaturexe2x80x9d Clark electrode has been described, but this electrode is a complicated multi-component part which must, also, be in contact with the solution to be measured. While an oxygen permeable membrane can be used to prevent the electrode components of the device from interacting with the constituents of the test solution, the oxygen must still equilibrate between the test solution and the measurement system and is consumed once it passes the membrane.
Optical systems which can yield oxygen concentration data, have been developed to overcome the shortcomings of the Clark electrode systems. The main advantage of such optical methods is that the instrumentation required to determine quantitative value does not itself make physical contact with the test solution. Optical techniques allowing both colorimetric and fluorometric analyses for oxygen to be carried out rapidly and reproducibly are known, and costs for such analyses are often quite low. For example, several luminescent techniques for the determination of oxygen have been described which are based on the ability of oxygen to quench the fluorescence or phosphorescence emissions of a variety of compounds. However, such methods have not been adapted to microbial monitoring or prokaryotic or eukaryotic cell monitoring.
Other systems have been described that provide information on the presence, identity and antimicrobic susceptibility of microorganisms in a period of eight hours or less. Wilkins and Stones in U.S. Pat. No. 4,200,493 disclose a system that uses electrodes and a high impedance potentiometer to determine the presence of microorganisms. In U.S. Pat. No. 3,907,646 Wilkins et al. disclose an analytical method which utilizes the pressure changes in the headspace over a flask associated with microbial growth for the detection and surveillance of the organisms. U.S. Pat. No. 4,220,715 to Ahnell, discloses a system wherein the head space gas above a test sample is passed through an external oxygen detector for determination of the presence of microorganisms. Ahnell, in U.S. Pat. No. 4,152,213, discloses a system for analysis by monitoring the vacuum produced by growing organisms in a closed head space above a test sample. U.S. Pat. No. 4,116,775 to Charles et al. is an example of the use of optical means based on the increase in turbidity or optical density of a growing microbial culture for the detection and monitoring of bacterial growth. A combined electro-optical measurement of birefringence of a test solution containing microorganisms is described in EPO 0092958 (Lowe and Meltzer).
The increased incidence of tuberculosis and the recent emergence of Multiple Drug Resistant (MDR) strains threatens the ability to control this disease. Therefore, when a strain is resistant to two or more drugs, such as rifampin and isoniazid, the course of treatment increases from 6 months to 24 months, and the cure rate decreases from almost 100% to less than 60%.
Mycobacterium tuberculosis (TB) is a slow growing species. Generally, at least three to five weeks of growth on solid or liquid media are required to produce enough cell mass for identification and susceptibility testing. The most commonly used susceptibility method for TB is the Modified Proportion Method (NCCLS M24-T). This method requires an additional three to four weeks of growth before the results are available. The total elapsed time for a find report is typically two months and may be as much as three months.
The BACTEC 460 instrument (Becton, Dickinson and Company, Franklin Lakes, N.J.) can reduce these times considerably. The BACTEC method detects the presence of mycobacteria by their production of radioactive CO2. The BACTEC system can also detect resistant organisms by their continuing production of radioactive CO2 in the presence of antimycobacterial drugs.
It becomes apparent that a wide variety of methods have been applied to the detection and the antibiotic susceptibility testing of microorganisms. Many of these methods can only yield useful data when monitored by instruments dedicated to this task. Thus there exists a need for a system which can allow determinations of the presence and behavior of microorganisms without the requirement of dedicated instrumentation. Further there exists a need for a system that will allow the determination of the effect of a compound such as an antibiotic on a sample of microorganisms in a short time that does not significantly alter the behavior of the microorganisms.
There also currently exists a need for improved methods of measuring eukaryotic and/or prokaryotic cell growth and viability, such as, for example, in the areas of drug discovery and development. An important application for these methods is in testing and quantifying the effects of therapeutic drugs, drug candidates, toxins and chemicals on the growth of cell lines (i.e, cytotoxicity assays). As an example, potential chemotherapeutic drug candidates are frequently tested at a number of concentrations to determine their potency for inhibiting the growth of selected mammalian tumor cell lines.
The most commonly used reagent for eukaryotic (i.e., mammalian) cell cytotoxicity assays is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [xe2x80x9cRapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assaysxe2x80x9d, T. Mossmann, J. Immunol. Methods (1983), vol. 65, 55-63]. This tetrazolium salt is reduced within the mitochondria of metabolically active cells to form a colored precipitate (formazan dye). For cytotoxicty measurements, the cells are typically grown in a microwell trays containing various concentrations of drug. MTT is added and incubated with cells for 1-4 hours, the cells are lysed, the formazan dye is resolubilized by thorough mixing and a dose-response curve is obtained from endpoint absorbance measurements. Among disadvantages of this method are the multiple reagent additions which are required. MTT is also susceptible to interferences from some drugs with reducing groups and from precipitation of some drugs, especially those adsorbing light in the visible region. The test itself is non-reversible and further time point readings of the same cell cultures cannot be performed without setting up a separate assay to be used for each time point.
Another redox indicator suggested for cytotoxicity assays is resazurin which is reduced to resorufin in the presence of growing cells. Resazurin is subject to autoreduction in some media which can cause false positive signals. An improved formulation of resazurin with a redox stabilizing buffer known as xe2x80x9cAlamar Bluexe2x80x9d has been introduced to solve this autoreduction problem in U.S. Pat. No. 5,501,959. This formulation, however, still requires the addition of dye and buffer to the cells and is essentially a non-reversible reduction.
Another method for determining cell viability is to measure uptake of radiolabeled nucleotides such as tritiated thymidine. This test is very sensitive but it is relatively expensive, time-consuming, and requires multiple steps. It also requires the handling and disposal of radioisotopic waste. This type of assay cannot readily be automated or adapted to formats for rapid drug screening purposes.
It is therefore an object of this invention to provide an improved means to detect the presence of, and to evaluate the metabolic activity of, eukaryotic cells present in a liquid or semi-solid media. It is further an object of this invention to provide a microbial monitoring device or system which can be simply read and visually interpreted, and which permits results to be obtained in a shorter time period than previously attainable, nominally 6 hours or less. Additionally, it is an object of the invention to provide a means for detection and/or monitoring the activity of eukaryotic cells without the use of dedicated instrumentation.
The above and related objects are realized by the processes of the instant invention. These processes utilize a luminescence detection system, and more particularly, a fluorescence detection system wherein the fluorescing sensor compound is one which exhibits a quantifiable degree of quenching when exposed to oxygen. In one embodiment, the sensor compound may be brought into contact with the test sample (either directly or separated by an oxygen permeable membrane) and the fluorescence is measured or observed visually with appropriate aids. In another embodiment, an increase in fluorescence is indicative of respiring aerobic microorganisms, which utilize (and thereby reduce) the oxygen in the sample.
The sensor need not be in direct contact with the test sample. The only requirement is that the test sample and sensor are in a container substantially isolated from atmospheric oxygen so that the sensor can react to the presence/absence of oxygen in the container.
The system can, thus, be used to detect a variety of respiring eukaryotic and/or prokaryotic cells and can be used in cytoxicity assays for the effects of drugs, toxins, or chemicals on eukaryotic and/or prokaryotic cells.